Nozzle and method for flow cytometry

ABSTRACT

The invention relates to a nozzle for flow cytometry, the housing of which is tapering towards an outlet and in which a feed tube is arranged for a core flow liquid, the outlet opening of which is arranged at a distance from the outlet of the housing. The outlet of the housing forms the outlet of the nozzle. The housing of the nozzle extends from its outlet, which is arranged at its first end to its opposite second end, and has an inlet for a sheath flow liquid connected with the internal volume. The nozzle is characterized in that in the housing a leading element that promotes the alignment of particles extends from both sides of the feed tube.

PRIORITY CLAIM AND REFERENCE TO RELATED APPLICATION

This application is a divisional application and claims priority under35 U.S.C. §§ 119, 120, 121 and 365(c) from prior co-pending applicationSer. No. 14/888,906, filed Feb. 3, 2016, which application isincorporated by reference herein and which application claimed priorityfrom PCT/EP2014/059505, filed May 8, 2014, which application claimedpriority from German Application 10 2013 208 584.2, filed May 8, 2013and EP 13168370.8, filed May 17, 2013.

FIELD-INTRODUCTION

The present invention relates to a nozzle for a flow cytometer, a flowcytometer with the nozzle and a method that can be carried out with thenozzle resp. with a flow cytometer having the nozzle, for alignment ofparticles in a liquid flow, which has a particle-containing core flowwithin a sheath flow or consists thereof. In particular, the inventionconcerns the use of the nozzle in a method for the alignment ofparticles and sorting of particles depending on a property that isdetected following the passage of particles through the nozzle. Here,sorting is preferably the deflection of sections of the fluid flow, inparticular of drops that are formed from the fluid flow, into at leasttwo fractions. In this embodiment, the invention relates to theproduction of a fraction of particles by alignment of the particles in aliquid flow with the nozzle according to the invention.

Preferably, the particles, which are aligned according to the method andare optionally sorted into fractions, have an altogether flat shape, andare symmetrical, for example, to a section plane, in such a manner thatthe particles have a cross-section with a first and a second dimensionthat is perpendicular to its longitudinal axis, wherein the crosssection in the first dimension is smaller than in the second dimension.Preferred particles are biological cells, such as blood cells, inparticular plate-shaped cells and non-human mammalian sperm, inparticular sperm derived from a male animal, which is in particular abovine, pig, sheep, elephant, camel, horse or a billy goat.

PRIOR ART

U.S. Pat. No. 6,149,867 describes a generic device and a method with anozzle for sorting of mammalian sperm in sex chromosome specific spermfractions. The method uses a flow cytometer with a nozzle, the housingof which tapers conically and in which a feed line for the core fluidcontaining sperm is contained, in such a manner that a core flowcontaining sperm flows out at the nozzle outlet, surrounded by a sheathflow.

WO 99/05504 describes a nozzle for use in a flow cytometer for sortingof sperm, which is characterized in that the housing of the nozzle inthe section between the outlet opening of a feed tube for core flowliquid containing sperm has an essentially funnel-shaped inner surface,which has a first elliptical cross-section, and an adjacent axialsection with elliptical cross-section, which is rotated by 90° withrespect to the elliptical cross-section of the upstream section.

EP 1238261 B1 describes a nozzle for a flow cytometer for sorting ofsperm, in which a feed tube for core flow liquid containing spermarranged coaxially in a nozzle runs from a section with a cylindricalouter diameter to an essentially rectangular cross-section within asection in the shape of a truncated cone of the nozzle, wherein atapering section of the nozzle with elliptical cross-section joinsdownstream of this inlet tube.

TASK OF THE INVENTION

With respect to the prior art, the task of the invention is to providean alternative nozzle for alignment of particles in a liquid flow and amethod for alignment of not rotationally symmetrical particles in aliquid flow. A preferred task is to provide a nozzle, which is easy tomanufacture, has in particular only components with inner surfaces,which are rotationally symmetrical and are therefore easy to produce.

GENERAL DESCRIPTION OF THE INVENTION

The invention solves the task with the features of the claims, inparticular with a nozzle, the housing of which is tapering towards anoutlet and in which a feed tube is arranged for a core flow liquid, theoutlet opening of which is arranged at a distance from the outlet of thehousing. The outlet of the housing forms the outlet of the nozzle. Thehousing of the nozzle extends from its outlet, which is arranged at itsfirst end opposite to its second end, and has an inlet for a sheath flowliquid associated with the internal volume. The second end of thehousing can be covered with a cover, in which the inlet for sheath flowliquid is optionally arranged. Preferably, the internal cross section ofthe housing is rotationally symmetrical resp. shaped in a circularmanner and is correspondingly formed by a rotationally symmetrical innersurface, which runs at least in sections in a conical manner to theoutlet. The feed tube arranged inside the housing is preferably arrangedcoaxially with respect to the longitudinal axis of the housing. Theinlet opening of the feed tube is connected to a feed line for aparticle-containing core flow liquid. The feed tube is preferably madeof metal.

The nozzle according to the invention is characterized in that a leadingelement extends in the housing on both sides of the feed tube with across section, which extends further in a first dimensionperpendicularly with respect to the longitudinal axis of the housingthan in a second dimension arranged perpendicularly with respect to thefirst dimension, which second dimension is also called the thickness ofthe leading element. The leading element, which extends in the nozzle onboth sides of the feed tube is beneficial to the alignment of particles,which are contained in the liquid flow, in particular in the core flow,in particular in such a manner that particles with a stretched crosssection are brought into a common alignment, preferably into analignment, in which the longer extension of the cross section of theparticles is arranged approximately parallel to the first dimension ofthe leading element. The feed tube may be arranged within the leadingelement or formed as a bore hole within the leading element. The leadingelement therefore extends in the first dimension up to a smaller spacingfrom the inner wall of the housing, while it is further spaced apartfrom the inner wall of the housing in the second dimension. The leadingelement ends up in an edge, which limits the leading element in itsfirst dimension and has the thickness of the second dimension. Theleading element forms with the inner wall of the housing a clearcross-section, which is separated into two portions that are spacedapart by the leading element and preferably contact each other resp.converge exclusively in the area of the clear cross-section by which theleading element is spaced apart from the inner wall of the housing inthe first dimension. The leading element has a thickness, which extendsin the second dimension of its cross section, which is smaller than itsextension along its first dimension. In the second dimension, theleading element can extend e.g. up to at maximum 70%, preferably atmaximum 50%, preferably at maximum 30%, at maximum 20% or at maximum 10%with respect to the extension in the first dimension. In the seconddimension, the cross-section of the leading element may change along thefirst dimension, in particular may decrease or increase along the firstdimension from the longitudinal axis of the housing to its edge.

Along the longitudinal axis of the housing, the leading element canextend in its first dimension up to the same spacing from thelongitudinal axis of the housing resp. up to the inner wall of thehousing, or extend with distances that are different along thelongitudinal axis of the housing, resp. up to a different distance fromthe inner wall of the housing. The cross section of the leading elementcan therefore end up in its first dimension in an edge, which isarranged along the longitudinal axis of the housing at the same distancefrom the inner wall of the housing, or along the longitudinal axis ofthe housing at a different distance from the inner wall of the housing.For example, the edge may be arranged in a section adjacent to the firstend of the leading element, generally also called first end section, ata smaller spacing from the inner wall of the housing than in an adjacentsection thereto, which is opposite the outlet opening of the feed tube.The edge can e.g. be arranged in the section adjacent to the first endof the leading element at an equal spacing along the longitudinal axisof the housing to the inner wall of the housing and, in the adjacentsection, which is opposite the outlet opening, at a spacing from theinner wall of the housing, which increases with increasing distance fromthe outlet opening. Optionally, the edge is rectilinear or arched,convex or concave in sections with respect to the longitudinal axis ofthe housing. With respect to the second dimension, the edge may be plan,concave or convex, in particular parallel to the inner wall of thehousing.

The leading element preferably has in its first end section adjacent toits first end an essentially parallel edge to the inner wall of thehousing. The first end section is in particular arranged in the taperingsection of the nozzle, which is adjacent to the outlet opening. Thisarrangement of the first end section in the tapering section of thehousing allows an effective alignment of flat cells, in particular ofnon-human mammalian sperm. The first end section of the leading elementhas with increasing distance from the first end of the leading element alarger extension in the first dimension, so that the edges of the firstend section run at a distance to the inner wall of the housingessentially parallel to the tapering, in particular conically convergingsection of the housing. The edges of the first end section may then bearched, in particular convex, and run at a distance to the tapering, inparticular conically converging section of the housing. The first endsection allows its arrangement in this tapering section of the nozzle.The first end section can have edges, which are at a distance from 1 to10%, e.g. 2 to 5% of the distance between the longitudinal axis of thehousing and/or of the feed tube and the housing. Adjacent to the firstend section and opposite its first end, the leading element has a secondend section, which tapers towards the second end. The leading elementcan taper in its second end section opposite the first end section,resp. at its second end, in such a manner that it flows out or ends atthe feed tube. In this embodiment, the feed tube forms the carrier resp.the connection between the cover of the housing and the leading element,so that only the feed tube bears the leading element. In thisembodiment, the feed tube forms a cylindrical carrier for leadingelement, which is spaced apart from the cover.

Between the first and the second end sections, the leading elementpreferably has its largest extension in the first dimension.

Optionally, the leading element has a narrow resp. sharp edge, resp. itssmallest extension in the second dimension along the first and secondend sections. A narrow resp. sharp edge can promote a laminar flowbetween the leading element and the inner wall of the nozzle.

The second end section can taper opposite the first end section up tothe feed tube, resp. flow out at the feed tube. The second end sectioncan be spaced at a distance of at least 20%, preferably at least 30%,more preferably at least 50% of the length of the feed tube within thehousing, resp. the length of the feed tube between a cover of thehousing and its first end, from the cover of the housing. In thisembodiment, the feed tube bears the leading element alone, whichpreferably has a circular inner and outer cross section resp. iscylindrical. This arrangement of the leading element at a distance tothe cover on the feed tube is preferred.

Alternatively, a transitional section may connect opposite the first endsection to the second end section, the outer cross section of whichtransitional section increases and to which a cylinder section isadjacent. Therein, the cylinder section can extend up to the cover andform an annular clear cross section with the housing. The cylindersection preferably has a larger outer diameter than the extension of thesecond end section adjacent to the transitional section in its firstdimension, so that the second end section forms a constriction in atleast one of first and second dimension with the transitional section.In particular in embodiments, in which the first end section, optionallyand preferably also the second end section, have a very stretched crosssection, e.g. when the extension in the second dimension is 10 to 30% orat most 20% or up to 15% of the extension in the first dimension, thetransitional section connects the external surfaces of the cylindersection with the ones of the leading element. The cylinder section cane.g. have an outer diameter, which is greater than the constriction andsmaller than the largest extension of the leading element in its firstdimension.

For the generation of a liquid flow with animal cells as particles in acore flow liquid, the inner wall of the housing may taper e.g. from adiameter of approx. 4 to 10 mm up to the outlet, which can have e.g. adiameter of 0.2 to 1 or to 0.5 mm. The leading element can extend e.g.along the longitudinal axis of the housing over 3 to 5 mm and in thefirst dimension from approximately 0.5 mm to 1.5 mm at the first end toapproximately 3 to 5 mm at most, while it extends in the seconddimension approximately by 0.5 to 1 mm on both sides of the longitudinalaxis, respectively.

The leading element is preferably symmetrical to the longitudinal axisof the housing and consists in particular in two parts, which extendwith their first dimensions in a common plane, in which the longitudinalaxis of the housing is arranged, wherein, more preferably, the crosssection of the leading element is formed symmetrically along thelongitudinal axis of the housing.

The leading element causes an alignment of the particles contained inthe core flow liquid according to their shape in the section between theoutlet opening of the feed tube and the outlet of the nozzle. Thisalignment of the particles is currently attributed to the fact that theextension of the leading element in its second dimension causesdifferent flow velocities of the sheath flow liquid, in particular overthe first dimension of the leading element. It is assumed that thesedifferent flow velocities lead to an alignment of the particles, whichthey essentially also have after outflow from the outlet of the nozzle.

The nozzle has the advantage that the inner wall resp. the cross sectionof its housing may be rotationally symmetrical and can therefore bemanufactured easily, e.g. by means of drilling. Correspondingly, thehousing tapers by the fact that the inner wall spans a cross sectionthat decreases towards the outlet and is in particular rotationallysymmetrical about the longitudinal axis of the housing. Preferably, thecross section that is spanned from the inner wall of the housing isconical in the area adjacent to the outlet. Optionally, the crosssection that is spanned from the inner wall of the housing can taperover the section, which extends between the outlet opening of the feedtube and the outlet of the housing, in particular converge conically.Optionally, the cross section that is spanned from the inner wall of thehousing can taper over the section, which extends over the sectionbetween the outlet opening of the feed tube and the second end of theleading element or up to the second end of the housing, in particularadjacent to the section, which extends between the outlet opening of thefeed tube and the outlet of the housing, in particular convergeconically, or have another form, e.g. be cylindrical. Also the outletcan be rotationally symmetrical, in particular a round bore hole.Preferably, the nozzle has at its first end an insert made of hardmaterial, e.g. ceramic or sapphire, in which a bore hole is formed as anoutlet. The formation of the clear cross section of the nozzle, whichcontrols the flow of the sheath flow liquid, takes place through theleading element arranged in the housing, wherein the cross section isproduced by shaping the external surface of the leading element, resp.through the different extension of the leading element in its first andsecond dimension, which are perpendicular to the longitudinal axis andperpendicular to each other. The nozzle has therefore the advantage thatthe non-rotationally symmetrical surface of the leading element can bemanufactured as external surface, while the inner wall of the housingcan be manufactured as a rotationally symmetrical surface.

The leading element extends along the longitudinal axis of the housingfrom its first end up to its opposite second end, wherein the first endis arranged adjacent to the outlet opening of the feed tube or at asmall distance further away from the outlet of the housing, as theoutlet opening of the feed tube, e.g. by up to 10%, preferably by up to5% or 2% of the distance from the outlet opening of the feed tube to theoutlet of the housing. Preferably, the first end is arranged in theplane, in which the outlet opening of the feed tube lies. The second endof the leading element can abut to the second end of the housing or canbe arranged at a distance from the second end of the housing, e.g. at adistance from 1 to 80%, preferably 10 to 50% of the extension of thehousing from its outlet to its second end or to the second end of theleading element.

Preferably, the leading element extends in its first and seconddimension perpendicular to the feed tube and along the longitudinal axisof the housing, wherein in particular the feed tube is arrangedcoaxially with respect to the longitudinal axis of the housing.

Preferably, the nozzle at the inlet opening of the feed tube has abuffer container for core flow liquid, into which a feed line forparticle-containing core flow liquid discharges. Such buffer containerincreases the proportion of particles, which are arranged by the housingin a predetermined alignment. This is currently attributed to the factthat a buffer container reduces flow effects from the feed line, whichcontinue into the feed tube. More preferably, the nozzle has anoscillation generator, which is attached to a wall of the buffercontainer for core flow liquid, which is arranged opposite the inletopening of the feed tube. The oscillation generator is preferably apiezoelectric element that is impingeable with electric voltage.Particularly preferably, the oscillation generator, e.g. thepiezoelectric element, is attached under pretension against the wall ofthe buffer chamber for core flow liquid, e.g. pressed against the wallof the buffer chamber by a cover arranged between the oscillationgenerator and the internal volume of the buffer chamber.

Preferably, the nozzle at the inlet opening of the nozzle for sheathflow liquid has a buffer container for sheath flow liquid, which isoptionally arranged adjacent to the buffer container for core flowliquid, e.g. between the buffer container for core flow liquid and thesecond end of the housing, wherein further optionally the feed tube isled through the buffer container for sheath flow liquid. A buffercontainer for sheath flow liquid reduces effects of the feed line ofsheath flow liquid to the flow of the sheath flow liquid in the clearcross section of the housing and increases the alignment of particlesinto a predetermined alignment.

The feed tube preferably has a circular internal cross section which cantaper along the longitudinal axis of the housing and is preferablyconstant along the longitudinal axis of the housing.

The piezoelectric element serves as an oscillation generator, whichpreferably generates pressure waves that run perpendicular to thelongitudinal axis of the housing, in order to generate a droplet flow incase of arrangement of the outlet of the housing in a gas-filled space.

A flow cytometer with the nozzle according to the invention preferablyhas at least one first radiation source, which is oriented towards afirst section of the liquid flow coming out of the housing of thenozzle, e.g. a laser, and a first detector oriented opposite theradiation source towards the first section of the liquid flow, whereinoptionally the detector generates a signal, which controls a deflectionapparatus in order to deflect sections of the liquid flow depending onthe detection by means of the signal, e.g. to fractionate. Optionally,the device has a second radiation source, which is oriented e.g. towardsa second section of the liquid flow between the first radiation sourceand the nozzle, and a second detector, which is oriented towards thissecond section. Preferably, the second detector generates a secondsignal, which controls the deflection apparatus, so that sections of theliquid flow are deflected additionally depending on the second signal.

The deflection apparatus can be a pair of electrically oppositelycharged plates, which are arranged on both sides of the liquid flow, andhave an electric contact, which is arranged in the nozzle, in particularin the housing. Optionally, the contact can be the feed tube for coreflow liquid. Preferably, the electric contact is controlled depending onthe first and/or second signal, so that positive or negative chargingtakes place depending on the first and/or second signal. Alternatively,the deflection apparatus can be a laser directed at the liquid flow,which is set up to evaporate the liquid flow only superficially up tosuperficial evaporation of the liquid flow, as is described e.g. inWO2010/149739.

The method according to the invention using the nozzle resp. a flowcytometer with the nozzle has the following steps:

-   -   providing a particle-containing core flow liquid,    -   providing a sheath flow liquid,    -   pumping the sheath flow liquid through the inlet opening for        sheath flow liquid,    -   pumping the particle-containing core flow liquid through the        inlet opening of the feed tube, wherein a leading element        extends along the longitudinal axis of the housing, wherein the        feed tube is preferably arranged coaxially with respect to the        longitudinal axis of the housing, wherein the leading element        divides the clear cross section of the internal volume of the        housing in two parts, and wherein the leading element extends        along the longitudinal axis to a greater degree along a first        dimension perpendicular to the longitudinal axis up to a        distance to the inner wall of the housing than it extends in a        second dimension perpendicular to the longitudinal axis and to        the first dimension,    -   flowing of sheath flow liquid through the clear cross section of        the housing and flowing of core flow liquid through the feed        tube, wherein the core flow liquid is contacted by the sheath        flow liquid after outflow from the outlet opening of the feed        tube and the particles contained in the core flow liquid are        moved into a predetermined alignment,    -   optionally allowing passage through a tapering section between        the outlet opening of the feed tube and the outlet of the        housing, whereby the cross section of the sheath flow liquid and        the core flow liquid is reduced, and    -   exiting of the core flow liquid surrounded by a sheath flow        liquid through the outlet of the housing.    -   Optionally, the method contains the step of detecting a property        of the particle and,    -   further optionally, the step of treating (e.g. through laser        irradiation) and/or of deflecting the particles into separated        fractions resp. containers depending on a detected property.    -   Preferably, the housing is rotationally symmetric.

In particular preferred is a method for preparingsex-chromosome-specifically sorted fractions of non-human sperm, inwhich the sperm is moved through the nozzle in a predetermined alignmentand is moved in this alignment before the radiation path of a detectorand detected.

Optionally, the leading element is slipped onto the feed tube, in apreferably reversible resp. releasable manner, and attached e.g. at thesecond end of the housing. Preferably, the leading element is formed inone piece with the feed tube and attached on one end opposite its outletopening, e.g. by means of engagement with a cover, which limits theinternal volume of the housing at its second end.

The feed tube may have a circular internal cross section. Alternatively,the feed tube may have a stretched internal cross section, e.g. anelliptical or rectangular internal cross section, e.g. with roundedinner edges, wherein the longer extension of a stretched internal crosssection is preferably arranged approximately parallel to the firstdimension. Preferably, the leading element has a symmetry plane, throughwhich the longitudinal axis runs, and particularly preferably theinternal cross section of the feed tube extends in a symmetry plane thatis shared with the leading element. A stretched internal cross sectionof the feed tube can e.g. have a ratio of the long to the shortextension of at most 0.3 or at most 0.2.

The nozzle, preferably with an insert made of ceramic or sapphire, whichforms the outlet at the first end of the housing, can consist ofplastics, e.g. made of PEEK or POM, optionally of ceramic. The feed tubecan consist of metal and the leading element consist of plastics, e.g.made of PEEK or POM; preferably, the feed tube is formed as a bore holewith a round cross section in the leading element, which consists ofplastics, e.g. made of PEEK or POM. Preferably, the method is a methodfor producing fractions of non-human mammalian sperm and has the stepof, after the flow of a core flow liquid containing the non-human spermof an individual through the feed tube of the nozzle, replacing the feedtube and the leading element by another feed tube and leading element orsterilizing it before core flow liquid containing the non-human sperm ofanother individual is allowed to flow through the feed tube of thenozzle.

The invention will now be described more precisely by means of examplesand with reference to the figures, which schematically show in

FIG. 1 a nozzle according to the invention in cross section along thelongitudinal axis of the nozzle,

FIG. 2 a cross section of the leading element arranged in the nozzleperpendicular to the longitudinal axis of the nozzle,

FIG. 3 a nozzle according to the invention without its cover and

FIG. 4 A)-D) leading elements.

In the figures, the same reference numerals designate and refer tofunctionally equivalent elements.

FIG. 1 shows a nozzle with a housing 1, which extends along alongitudinal axis 5 from a round outlet 2 at its first end 3 to itsopposite second end 4. The second end 4 is covered by a cover 6, throughwhich the feed tube 7 extends coaxially with respect to the longitudinalaxis 5. The feed tube 7 discharges at its first end 8 into an outletopening 9 and at its opposite second end 10 has an inlet opening 11 fora particle-containing core flow liquid. The leading element 12 a, 12 bis arranged at a distance to the cover 6, which closes the housing 1 atits first end 4, and is carried by the feed tube 7.

FIG. 1 shows the leading element 12 a, 12 b in different designs, whichare represented respectively in halves on both sides of the feed tube 7,so that a design respectively extends symmetrically with respect to thelongitudinal axis 5. The leading element 12 a, 12 b extends from itsfirst end 13 a, 13 b, which is spaced apart from the first end 8 of thefeed tube 7, to its second end 14 a, 14 b. By way of example with theleading element 12 b, it is shown according to the preferred embodimentthat its first end 13 b can be arranged at the first end 8 of the feedtube 7 and can end flush with it. The second end 14 b of the alternativeleading element 12 b shows that a leading element 12 a, 12 b can extendto a different extent along the longitudinal axis 5 of the housing 1,e.g. can be arranged at a distance farther from the outlet of thehousing 1 than the outlet opening 9 or that the leading element 12 b canproject over the feed tube 7, as is shown at the first end 13 b, or e.g.that its first end 13 b′ is arranged by a smaller distance farther fromthe outlet of the housing. The schematically drawn cross sections 15 a,15 b of the leading element 12 a, 12 b extend perpendicular to thelongitudinal axis 5 in a first dimension 16, toward which the thicknessof the leading element 12 a, 12 b extends in the second dimension 17perpendicular thereto. As shown by way of example with the cross section15 a, the leading element 12 a may have a constant thickness in thesecond dimension 17, or as shown by way of example with the crosssection 15 b, the leading element 12 b may have a cross section 15 b,which changes in the second dimension 17 along the first dimension 16,e.g. decreases with increasing distance to the longitudinal axis 5.

It is shown in FIG. 1 that the leading element 12 a, 12 b extends inparticular in a section, which is adjacent to its first end 13 a, 13 balong the first dimension 16 of the longitudinal axis 5 up to anapproximately constant distance from the inner surface of the housing 1,resp. extends up to an edge 18 at an approximately constant distancefrom the inner surface of the housing 1.

The first end section 25, which is adjacent to the first end 13 a, 13 bof the leading element 12 a, 12 b, of the leading element 12 a, 12 b isarranged within the section of the housing 1 that conically convergestowards the outlet 2. The second end section 26 is adjacent opposite thefirst end 13 a, 13 b of the leading element 12 a, 12 b to the first endsection 25, wherein the leading element has its largest extension 29 inthe first dimension, where the first and second end section 25, 26 areadjacent to each other. The design of the leading element 12 a, 12 b,which is such that its first end section 25 is arranged within theconically converging section of the housing 1, allows an effectivealignment of cells with a flat shape, e.g. of non-human mammalian sperm,for their subsequent sorting.

FIG. 1 shows that the edge 18 of the first end section 25 runsessentially parallel and at a distance to the inner wall of the housing1 and therein can be formed in a slightly convex manner. In general, inthis embodiment, the leading element 12 a, 12 b can be slipped onto thefeed tube 7, optionally clamped or arranged by means of a snap lock or athread on the feed tube 7.

According to the preferred embodiment, FIG. 1 shows a nozzle with abuffer container 19 for core flow liquid, into which a supply line 20for core flow liquid runs and to which the inlet opening 11 of the feedtube 7 is connected. A piezoelectric element 21 is attached underpretension in the buffer container 19 for core flow liquid opposite theinlet opening 11 of the feed tube 7. Furthermore, a buffer container 22for sheath flow liquid is connected to the inlet 23 for sheath flowliquid, which discharge into the housing 1, wherein the buffer container22 for sheath flow liquid is connected to a supply line 24 for sheathflow liquid. The buffer container 22 for sheath flow liquid is arrangedbetween the second end 4 of the nozzle 1 and the buffer container 19 forcore flow liquid, wherein the feed tube 7 is arranged through the buffercontainer 22 for sheath flow liquid.

FIG. 2 shows cross sections 15 a, 15 b through the leading element 12 a,12 b that are perpendicular to the longitudinal axis 5 of the nozzleresp. of the housing 1, wherein only half of the leading element 12 a,12 b, which extends mirror-symmetrically towards the second dimension 17resp. to the longitudinal axis 5. Preferably, the feed tube 7 isarranged in a circular manner about the longitudinal axis 5 of thehousing 1, which lies in the intersection of the first dimension 16 withthe second dimension 17. As is generally preferred, the outlet 2 of thehousing 1 is arranged symmetrically with respect to the longitudinalaxis 5 of the housing 1.

FIG. 3 shows a nozzle, in the housing 1 of which a leading element 12 ais arranged, in which the feed tube 7 is carried out as a bore hole,which is arranged coaxially with respect to the longitudinal axis 5 ofthe housing 1. The outlet 2 of the housing 1 is formed by an insert 1 a,which is shaped as a truncated cone in a section adjacent to the outlet2 and, adjacent thereto, cylindrical. The first end section 25 is, as isgenerally preferred, arranged within the tapering section of the housing1. The leading element 12 a, 12 b in the area adjacent to its second endsection 26 has a transitional section 27, to which in turn a cylindersection 28 is adjacent. The cylinder section 28 extends across theentire length of the feed tube 7. The transitional section 27 forms aconstriction 30 with the second end section 26 of the leading element12.

FIG. 4A) shows the leading element 12 a of FIG. 3 in a reducedrepresentation rotated by 90° about the longitudinal axis 5. It isclearly visible in FIGS. 3 and 4A) that the leading element 12 a extendsfarther in the first dimension 16 that is perpendicular to thelongitudinal axis 5 than in the second dimension 17 that isperpendicular to the longitudinal axis 5 and to the first dimension 16.

FIG. 4B) shows an optional embodiment of a leading element 12 a, whichextends in a section that is adjacent to the outlet opening 9considerably farther in its first dimension 16 than in its seconddimension 17, which is perpendicular to the image plane, while the crosssection of the leading element 12 a is tapered in the first dimension 16in an adjacent section, which is spaced apart from the outlet opening 9.

FIG. 4C) shows a cross section 15 a of a leading element 12 a, which isperpendicular to the longitudinal axis 5 of the housing, in which thefeed tube 7 is formed as a central bore hole with a round cross section.

FIG. 4D) shows a leading element according to FIG. 4C), in which thefeed tube 7 has an elliptical internal cross section, the long extensionof which is parallel to the first dimension 16 and the short extensionof which is parallel to the second dimension 17. Moreover, FIG. 4D)shows that the internal cross section of the feed tube 7 has the samesymmetry plane that runs through the longitudinal axis (perpendicular tothe representation plane) as the cross section of the leading element 12a.

Example 1: Detection of Y-Chromosome-Containing Sperm in Fresh Semen andSex-Specific Sorting

Freshly obtained bull semen was diluted in the usual manner in a diluentand incubated with a DNA-specific dye, e.g. Bisbenzimid H 33342(Hoechst), for 30 to 60 min at a temperature of 20° C. to 40° C. andsubsequently irradiated in a flow cytometer according to U.S. Pat. No.5,125,759 or DE 10 2005 044 530 with light with the appropriateexcitation wavelength for the dye. The respective emission was measured.

The alignment of the sperm was determined with a detector, which wasoriented directly downstream the nozzle towards the exiting liquid flowconsisting of individual droplets. The total DNA-content was determinedwith a further detector, which was oriented further downstream towardsthe liquid flow. The deflection apparatus had two oppositely chargedplates on both sides of the liquid flow and a contact for electricallycharging the liquid in the nozzle. This charge was fed, as is known,depending on the signal of the detector, which determines the alignmentof the sperm, and the polarity of the charge depending on the signal ofthe detector determining the total DNA-content. In this manner, thespermatozoa were deflected depending on the detected signal through anelectric field into sex-chromosome-specific fractions.

Optionally, a fluoride was added for immobilization of the sperm, e.g.into the sheath liquid or transport liquid used in the course of thesorting process, and/or before or during the addition of the dye inorder to increase the penetration of the dye into the spermatozoa.Fluoride ions were added in the range of 0.1 to 100 mM, preferably of 10nM to 10 mM. It was found that the optimal concentration of thefluoride, e.g. NaF or KF, diverged between different species and forindividuals. The optimal concentration for the species is specific andcould generally be determined as the concentration, which in themicroscopic analysis resulted in an immobilization of at least 90% ofthe spermatozoa, preferably of essentially all spermatozoa. Accordingly,the present invention also refers to compositions of the sperm fractionsprepared by the method according to the invention, and to methods forpreparing sex-specific sperm fractions and subsequently preserving thesperm fractions of non-human mammals, each preferably in the presence offluoride and/or anti-oxidants.

LIST OF REFERENCE SIGNS

-   1 housing-   1 a insert-   2 outlet-   3 first end of the housing-   4 second end of the housing-   5 longitudinal axis-   6 cover-   7 feed tube-   8 first end of the feed tube-   9 outlet opening-   10 second end of the feed tube-   11 inlet opening-   12 a, 12 b leading element-   13 a, 13 b, 13 b′ first end of the leading element-   14, 14 a, 14 b second end of the leading element-   15 a, 15 b cross-section of the leading element-   16 first dimension-   17 second dimension-   18 edge-   19 buffer container for core flow liquid-   20 supply line for core flow liquid-   21 piezoelectric element-   22 buffer container for sheath flow liquid-   23 inlet for sheath flow liquid-   24 supply line for sheath flow liquid-   25 first end section-   26 second end section-   27 transitional section-   28 cylinder section-   29 largest extension in first dimension-   30 constriction

The invention claimed is:
 1. A method for producing a liquid flow havinga particle-containing core flow surrounded by a sheath flow byintroducing a particle-containing core flow liquid into an inlet openingfor core flow liquid and introducing a sheath flow liquid into an inletopening for sheath flow liquid of a nozzle, wherein the sheath flow isgenerated by a leading element that extends along a longitudinal axis toa larger degree along a first dimension perpendicular to thelongitudinal axis up to a distance away from the inner wall of a housingthan it extends in a second dimension that is perpendicular to thelongitudinal axis and perpendicular to the first dimension, wherein theleading element extends from a first end, which lies in the plane of thefirst end of a feed tube or is offset by a distance from the first endof the feed tube, up to its second end, which is arranged at a distancefrom the second end of the nozzle, wherein the leading element in thenozzle extends on both sides of the feed tube for aligning the particleswhich are contained in the core flow, wherein the extension of theleading element in its second dimension causes different flow velocitiesof the sheath flow liquid, which different flow velocities lead to analignment of the particles, and wherein the particles have the alignmentalso after outflow from the outlet of the nozzle.
 2. The methodaccording to claim 1, wherein the particles have a cross-section, whichis smaller in a first dimension that is perpendicular to thelongitudinal axis of the housing of the nozzle than in a seconddimension.
 3. The method according to claim 1, wherein at least oneproperty of the particles contained in the core flow is detected in theliquid flow exiting the nozzle and the particles are separated into atleast two fractions depending on the detected property, or are treateddifferently depending on the detected property.
 4. The method accordingto claim 1 for the production of a preparation of particles, wherein theparticles are non-human mammalian sperms, the detected property is thepresence of the X chromosome or Y chromosome within the sperm, and bythe irradiation of the sperm depending on the detected property ordeflection of the sperm depending on the detected property into at leasttwo different fractions.
 5. The method of claim 1, using the nozzle fora flow cytometer, wherein the housing has an internal cross-section thattapers along its longitudinal axis from its second end to its first end,and wherein, for a core flow liquid, the feed tube has a circularinternal cross-section, wherein the leading element extends from itsfirst end, which lies in the plane of the first end of the feed tube oris offset by a distance from the first end of the feed tube, up to itssecond end, which is arranged at a distance from the second end of thenozzle.
 6. The method of claim 1, wherein the extension of the leadingelement in its second dimension causes different flow velocities of thesheath flow liquid over the first dimension of the leading element. 7.The method of claim 1, wherein the leading element extends in the firstdimension up to a smaller spacing from the inner wall of the housing,while it is further spaced apart from the inner wall of the housing inthe second dimension, and the leading element ends in an edge, whichlimits the leading element in its first dimension and has the thicknessof the second dimension, and the leading element forms with the innerwall of the housing a clear cross-section, which is separated into twoportions that are spaced apart by the leading element and convergeexclusively in the area of the clear cross-section by which the leadingelement is spaced apart from the inner wall of the housing in the firstdimension.
 8. The method of claim 1, wherein the leading element has anarrow edge along a first end section and a second end section, whichnarrow edge promotes a laminar flow between the leading element and theinner wall of the nozzle.
 9. The method of claim 1 for preparingsex-chromosome-specifically sorted fractions of non-human sperm, inwhich the sperm is moved through the nozzle in a predetermined alignmentand is moved in this alignment to the radiation path of a detector anddetected.
 10. A method for producing a liquid flow having aparticle-containing core flow surrounded by a sheath flow by introducinga particle-containing core flow liquid into an inlet opening for coreflow liquid and introducing a sheath flow liquid into an inlet openingfor sheath flow liquid of a nozzle, wherein the sheath flow is generatedby a leading element that extends along a longitudinal axis to a largerdegree along a first dimension perpendicular to the longitudinal axis upto a distance away from the inner wall of a housing than it extends in asecond dimension that is perpendicular to the longitudinal axis andperpendicular to the first dimension, wherein the leading elementextends from a first end, which lies in the plane of the first end of afeed tube or is offset by a distance from the first end of the feedtube, up to its second end, which is arranged at a distance from thesecond end of the nozzle, wherein the leading element in the nozzleextends on both sides of the feed tube for aligning the particles whichare contained in the core flow, such that particles with a stretchedcross section are brought into a common alignment and wherein theparticles have the alignment also after outflow from the outlet of thenozzle.
 11. The method of claim 10, wherein the particles are alignedinto an alignment in which a longer extension of the cross section ofthe particles is arranged approximately parallel to the first dimensionof the leading element.
 12. The method of claim 10, wherein the leadingelement extends in the first dimension up to a smaller spacing from theinner wall of the housing, while it is further spaced apart from theinner wall of the housing in the second dimension, and the leadingelement ends up in an edge, which limits the leading element in itsfirst dimension and has the thickness of the second dimension, and theleading element forms with the inner wall of the housing a clearcross-section, which is separated into two portions that are spacedapart by the leading element and converge exclusively in the area of theclear cross-section by which the leading element is spaced apart fromthe inner wall of the housing in the first dimension.
 13. The method ofclaim 10 for preparing sex-chromosome-specifically sorted fractions ofnon-human sperm, in which the sperm is moved through the nozzle in apredetermined alignment and is moved in this alignment to the radiationpath of a detector and detected.
 14. The method of claim 10, wherein theextension of the leading element in its second dimension causesdifferent flow velocities of the sheath flow liquid over the firstdimension of the leading element.